Ribonucleoproteins (RNPs) perform essential biological functions such as transcription regulation, messenger RNA splicing, RNA processing, and translation. One of the most abundant regulatory RNPs in higher eukaryotes is the 7SK RNP, comprised of the long noncoding 7SK RNA and protein components that dynamically assemble to sequester and inactivate the transcription elongation activator P-TEFb. Release of P-TEFb, facilitated by recruitment of multiple protein factors to the 7SK RNP and conformational changes in 7SK RNA, enables RNA Polymerase II processive transcription elongation. Dysfunction is linked to multiple diseases including heart disease, several cancers, and developmental disorders. Furthermore, IV exploits the 7SK RNP regulatory pathway to assist in its own replication. Despite its significance, information on the 7SK RNP assembly, structure, and mechanism of action remains extremely limited, which could guide the development of therapeutic agents using a structure-based approach. Information is particularly lacking on the central 7SK RNA stem-loop , which has an integral role in P-TEFb disassembly by recruiting proteins that promote P-TEFb release. Our long-term goal is to identify the mechanism of P-TEFb assembly onto the 7SK RNP and disassembly from the 7SK RNP. Toward this goal, we propose to work on two aims: (1) characterize 7SK RNA and protein subunits required for P-TEFb release. This approach will use a combination of solution NMR spectroscopy, binding assays, and X-ray crystallography to inform on the structural properties of this integral RNA element and provide insights into how this domain recruits specific proteins that then promote P-TEFb dissociation. (2) We will generate a stable cell line and develop a purification strategy to isolate the 7SK RNP from the EK T mammalian cell line to determine high- resolution structures of the 7SK-P-TEFb RNP and 7SK RNP after P-TEFb release. To accomplish this aim, we will apply innovative approaches to comprehensively characterize the architecture of the 7SK RNP. y interrogating the biophysical properties governing RNP assembly and stability we will achieve a rigorous understanding of how the 7SK RNP regulate transcription in healthy individuals, and how dysfunction leads to divergent disease phenotypes. Our experimental strategy is grounded in the hypothesis that characterizing the structure and assembly of the 7SK RNP will enable an understanding into the mechanism of transcription inactivation (P-TEFb assembly) and productive transcription elongation (P-TEFb dissociation), future studies to probe the mechanism further in vivo from this knowledge, and rational design of therapeutics to target the 7SK RNP. We will use highly interdisciplinary structure-based approach to investigate mechanisms of protein-RNA recognition as well as protein cooperactivity and competition in binding to structured RNA. These landmark studies will provide some of the first insights into the structural properties of 7SK RNA and protein elements required for P-TEFb release and re-activation.